Single (SSB) and double strand breaks (DSB) in supercoiled plasmid DNA pBR322 reacted with linoleic acid hydroperoxides (LOOH) were followed by agarose gel electrophoresis to obtain definitive information about factors affecting LOOH interaction with DNA. In water, LOOH induced extensive DSB, which were metal mediated and increased with incubation time. Adventitious metal bound to DNA was sufficient to decompose LOOH to reactive radicals, activity that was not readily inhibited by chelators DTPA and desferrioxamine. Added Fe2+ and Fe3+ increased SSB and DSB, although the effects of Fe2+ were more extensive. Above 100 microM both valences inhibited DNA damage. Strand breakage by LOOH proceeded via lipid alkoxyl and peroxyl radicals. Aldehydic lipid peroxidation products induced strand breaks via oxidation of double bonds, not by reactions of the carbonyl groups. Lipophilic antioxidants BHA, BHT, and alpha-tocopherol were about 20 times more effective than hydrophilic free radical scavengers sodium benzoate, inositol, DMSO, and mannitol in preventing LOOH-induced strand breaks, supporting lipid phase localization of the damage.